The release of neurotransmitters at synapses is responsible for signal transmission through brain circuits. Further, modifications in neurotransmitter release are likely to be involved in changing neuronal function during development, learning, disease states, and other forms of brain plasticity. The general goal of this project is to understand the molecular basis for the release of synaptic neurotransmitters. Many proteins have been identified in presynaptic terminals and this project seeks to clarify the function of a subset of these proteins that have been suggested to play key roles in the release of neurotransmitters. We will determine the possible role of synapsin in keeping synaptic vesicles in a reserve pool, as well as possible additional roles for this protein in the fusion of vesicles and their subsequent endocytotic recovery. Photochemical methods, such as photolysis of a caged peptide, will be used to determine whether the ATPase, NSF, is required before or after synaptic vesicles fuse during neurotransmitter release. Very similar methods will be used to determine the temporal order in which SNARE proteins and their binding partners interact during neurotransmitter release. Biochemical and physiological methods will be used to further test the hypothesis that the protein synaphin/complexin organizes SNARE proteins into a ring that is necessary for calcium-dependent triggering of neurotransmitter release. Finally, we will determine the possible roles of Eps15 and auxilin in the coating and uncoating of vesicles during endocytosis that follows neurotransmitter release. These experiments will be performed on the unique "giant" presynaptic terminal of squid, one of only a few synapses whose presynaptic terminals are sufficiently large to allow molecular probes to be microinjected. These experiments should clarify several important aspects of the molecular basis of synaptic communication in the brain and, thereby, ultimately yield insights into the etiology of numerous neurological disorders that result from defects in synaptic transmission.